PROJECT SUMMARY/ABSTRACT: Human botulism can result from the ingestion of food contaminated with botulinum neurotoxin (BoNT), from the infection of wounds or intestine, or from direct inhalation of the toxin, which can be transcytosed across epithelial barriers as a physiologically active molecule. Intoxication, if untreated, may lead to death due to respiratory muscle failure, while recovery from botulism is a prolonged process (up to several months) and BoNTs are classified as one of the six highest?risk threat agents for bioterrorism by the CDC. Developing may require intensive and expensive hospital care. Due to their extreme toxicity and ease of production, antidotes to botulinum neurotoxins is a priority goal of the Biodefense Research Agenda. During the previous funding period, we developed a molecular engineering platform that takes advantage of structural features and trafficking of BoNTs to enable the delivery within the presynaptic compartment of intoxicated neurons of single domain camelid antibodies (sdAbs) that bind and inactivate the wt light chain (LC) of BoNT metalloproteases. We developed a post-exposure anti-botulinum therapeutic (PEABT) targeting wt BoNT/A1, which is the first compound ever to show reversal of respiratory symptoms in mice at times post-intoxication when standard antibodies are ineffective. Preliminary data also demonstrated the effectiveness of a second lead candidate against wt BoNT/B, and attest to the generalizability of the platform. We succeeded in developing a novel principle for delivering single domain antibodies to the presynaptic compartment of neurons without a viral vector. In addition, we developed a murine model of botulism that is specifically appropriate to test botulism antidotes with an intra-neuronal mode of action. The murine model is currently being translated to a guinea pig, in response to an FDA suggestion that the guinea pig was the species to use for an Animal Rule pivotal study. Our objective is to expand the therapeutic range of this platform to two other known human pathogenic serotypes ? wt BoNT/B and wt BoNT/E, and to further increase the potency and therapeutic window of the wt BoNT/A1 countermeasure. We will identify sdAbs that bind and broadly neutralize BoNT/A, /B, and /E LC subtypes, and we will pursue strategies to increase the potency of PEABTs by increasing the affinity of sdAbs for the wt LC target and by improving therapeutic efficacy. The team of scientists assembled for this project includes researchers with unique expertise, who are well-qualified for the task at hand, including a long and successful history in generating BoNT-neutralizing sdAbs, extensive expertise in expressing neuron-targeted physiologically active fusion proteins that use metalloprotease-inactivated BoNT/C1 as a molecular vehicle for delivery of sdAbs, expertise in test systems for development of compounds promoting accelerated degradation of intracellular targets through the endogenous proteasomal system, and extensive experience in developing in vitro and in vivo models that can be used to test novel botulism antidotes.